The Conserved Cysteine-Rich Secretory Protein MaCFEM85 Interacts with MsWAK16 to Activate Plant Defenses.

Metarhizium anisopliae is an entomopathogenic fungus which may enhance plant growth and resistance when acting as an endophyte in host plants. However, little is known about the protein interactions nor their activating mechanisms. Common in fungal extracellular membrane (CFEM) proteins have been identified as plant immune regulators that suppress or activate plant resistance responses. Here, we identified a CFEM domain-containing protein, MaCFEM85, which was mainly localized in the plasma membrane. Yeast two-hybrid (Y2H), glutathione-S-transferase (GST) pull-down, and bimolecular fluorescence complementation assays demonstrated that MaCFEM85 interacted with the extracellular domain of a Medicago sativa (alfalfa) membrane protein, MsWAK16. Gene expression analyses showed that MaCFEM85 and MsWAK16 were significantly upregulated in M. anisopliae and M. sativa, respectively, from 12 to 60 h after co-inoculation. Additional yeast two-hybrid assays and amino acid site-specific mutation indicated that the CFEM domain and 52th cysteine specifically were required for the interaction of MaCFEM85 with MsWAK16. Defense function assays showed that JA was up-regulated, but Botrytis cinerea lesion size and Myzus persicae reproduction were suppressed by transient expression of MaCFEM85 and MsWAK16 in the model host plant Nicotiana benthamiana. Collectively, these results provide novel insights into the molecular mechanisms underlying interactions of M. anisopliae with host plants.

Inductive Production of the Iron-Chelating 2-Pyridones Benefits the Producing Fungus To Compete for Diverse Niches.

Diverse 2-pyridone alkaloids have been identified with an array of biological and pharmaceutical activities, including the development of drugs. However, the biosynthetic regulation and chemical ecology of 2-pyridones remain largely elusive. Here, we report the inductive activation of the silent polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) (tenS) gene cluster for the biosynthesis of the tenellin-type 2-pyridones in the insect-pathogenic fungus Beauveria bassiana when cocultured with its natural competitor fungus Metarhizium robertsii. A pathway-specific transcription factor, tenR, was identified, and the overexpression of tenR well expanded the biosynthetic mechanism of 15-hydroxytenellin (15-HT) and its derivatives. In particular, a tandemly linked glycosyltransferase-methyltransferase gene pair located outside the tenS gene cluster was verified to mediate the rare and site-specific methylglucosylation of 15-HT at its N-OH residue. It was evident that both tenellin and 15-HT can chelate iron, which could benefit B. bassiana to outcompete M. robertsii in cocultures and to adapt to iron-replete and -depleted conditions. Relative to the wild-type strain, the deletion of tenS had no obvious negative effect on fungal virulence, but the overexpression of tenR could substantially increase fungal pathogenicity toward insect hosts. The results of this study well advance the understanding of the biosynthetic machinery and chemical ecology of 2-pyridones. IMPORTANCE Different 2-pyridones have been identified, with multiple biological activities but unclear chemical ecology. We found that the silent tenS gene cluster was activated in the insect pathogen Beauveria bassiana when the fungus was cocultured with its natural competitor Metarhizium robertsii. It was established that the gene cluster is regulated by a pathway-specific regulator, tenR, and the overexpression of this transcription factor expanded the biosynthetic machinery of the tenellin 2-pyridones. It was also found that the paired genes located outside the tenS cluster contribute to the site-specific methylglucosylation of the main compound 15-hydroxytenellin. Both tenellin and 15-hydroxytenellin can chelate and sequester iron to benefit the producing fungus to compete for different niches. This study well advances the biosynthetic mechanism and chemical ecology of 2-pyridones.

A M35 family metalloprotease is required for fungal virulence against insects by inactivating host prophenoloxidases and beyond.

A diverse family of metalloproteases (MPs) is distributed in eukaryotes. However, the functions of MPs are still understudied. We report that seven MPs belonging to the M35 family are encoded in the genome of the insect pathogenic fungus Metarhizium robertsii. By gene deletions and insect bioassays, we found that one of the M35-family MPs, i.e. MrM35-4, is required for fungal virulence against insect hosts. MrM35-4 is a secretable enzyme and shows a proteolytic activity implicated in facilitating fungal penetration of insect cuticles. After gene rescue and overexpression, insect bioassays indicated that MrM35-4 contributes to inhibiting insect cuticular and hemocyte melanization activities. Enzymatic cleavage assays revealed that the recombinant prophenoloxidases PPO1 and PPO2 of Drosophila melanogaster could be clipped by MrM35-4 in a manner differing from a serine protease that can activate PPO activities. In addition, it was found that MrM35-4 is involved in suppressing antifungal gene expression in insects. Consistent with the evident apoptogenic effect of MrM35-4 on host cells, we found that the PPO mutant flies differentially succumbed to the infections of the wild-type and mutant strains of M. robertsii. Thus, MrM35-4 plays a multifaceted role beyond targeting PPOs during fungus-insect interactions, which represents a previously unsuspected strategy employed by Metarhizium to outmaneuver insect immune defenses.

Polyketides and a Dihydroquinolone Alkaloid from a Marine-Derived Strain of the Fungus Metarhizium marquandii.

Three new natural products (1-3), including two butenolide derivatives (1 and 2) and one dihydroquinolone derivative (3), together with nine known natural products were isolated from a marine-derived strain of the fungus Metarhizium marquandii. The structures of the new compounds were unambiguously deduced by spectroscopic means including HRESIMS and 1D/2D NMR spectroscopy, ECD, VCD, OR measurements, and calculations. The absolute configuration of marqualide (1) was determined by a combination of modified Mosher's method with TDDFT-ECD calculations at different levels, which revealed the importance of intramolecular hydrogen bonding in determining the ECD features. The (3R,4R) absolute configuration of aflaquinolone I (3), determined by OR, ECD, and VCD calculations, was found to be opposite of the (3S,4S) absolute configuration of the related aflaquinolones A-G, suggesting that the fungus M. marquandii produces aflaquinolone I with a different configuration (chiral switching). The absolute configuration of the known natural product terrestric acid hydrate (4) was likewise determined for the first time in this study. TDDFT-ECD calculations allowed determination of the absolute configuration of its chirality center remote from the stereogenic unsaturated γ-lactone chromophore. ECD calculations aided by solvent models revealed the importance of intramolecular hydrogen bond networks in stabilizing conformers and determining relationships between ECD transitions and absolute configurations.

Diverse effect of phosphatidylcholine biosynthetic genes on phospholipid homeostasis, cell autophagy and fungal developments in Metarhizium robertsii.

Phosphatidylcholine (PC) plays an important role in maintaining membrane integrity and functionality. In this study, two key genes (Mrpct and Mrpem) putatively involved in the cytidine diphosphate (CDP)-choline and phosphatidylethanolamine N-methyltransferase (PEMT) pathways for PC biosynthesis were characterized in the insect pathogenic fungus Metarhizium robertsii. The results indicated that disruption of Mrpct did not lead to any reduction of total PC content but impaired fungal virulence and increased cellular accumulation of triacylglycerol. Deletion of Mrpem reduced PC content and impaired fungal conidiation and infection structure differentiation but did not result in virulence defects. Lipidomic analysis revealed that deletion of Mrpct and Mrpem resulted in dissimilar effects on increase and decrease of PC moieties and other phospholipid species accumulations. Interestingly, we found that these two genes played opposite roles in activation of cell autophagy when the fungi were grown in a nutrient-rich medium. The connection between PC metabolism and autophagy was confirmed because PC content was drastically reduced in Mratg8Δ and that the addition of PC could rescue null mutant sporulation defect. The results of this study facilitate the understanding of PC metabolism on fungal physiology.

The Biosynthesis Pathway of Swainsonine, a New Anticancer Drug from Three Endophytic Fungi.

Swainsonine (SW) is the principal toxic ingredient of locoweed plants that causes locoism characterized by a disorder of the nervous system. It has also received widespread attention in the medical field for its beneficial anticancer and antitumor activities. Endophytic fungi, Alternaria sect. Undifilum oxytropis isolated from locoweeds, the plant pathogen Slafractonia leguminicola, and the insect pathogen Metarhizium anisopliae, produce swainsonine. Acquired SW by biofermentation has a certain foreground and research value. This paper mainly summarizes the local and foreign literature published thus far on the swainsonine biosynthesis pathway, and speculates on the possible regulatory enzymes involved in the synthesis pathway within these three fungi in order to provide a new reference for research on swainsonine biosynthesis by endophytic fungi.

Estrogen-mediated protection of the organotin-degrading strain Metarhizium robertsii against oxidative stress promoted by monobutyltin.

Dibutyltin (DBT) is a global pollutant characterized by pro-oxidative properties. The fungal strain Metarhizium robertsii can eliminate high levels of DBT efficiently. In this study, induction of oxidative stress as well as its alleviation through the application of natural estrogens during the elimination of DBT by M. robertsii were evaluated. During the first 24 h of incubation, the initial concentration of DBT (20 mg l-1) was reduced to 3.1 mg l-1, with simultaneous formation of a major byproduct - monobutyltin (MBT). In the presence of estrone (E1) or 17ß-estradiol (E2), the amounts of dibutyltin residues in the fungal cultures were found to be approximately 2-fold higher compared to cultures without estrogens, which was associated with the simultaneous utilization of the compounds by cytochrome P450 enzymes. On the other hand, MBT levels were approximately 2.5 times lower in the fungal cultures with the addition of one of the estrogens. MBT (not DBT) promotes the generation of O2-, H2O2, and NO at levels 65.89 ± 18.08, 4.04 ± 3.62, and 27.92 ± 1.95, respectively. Superoxide dismutase and catalase activities did not show any response of the M. robertsii strain against the overproduction of superoxide anion and hydrogen peroxide. Application of E1 as well as E2 ensured non-enzymatic defense against nitrosative and oxidative stress through scavenging of nitrogen and oxygen reactive species, and limited their levels from 1.5-fold to 21-fold, depending on the used estrogen.

Disruptions of the genes involved in lysine biosynthesis, iron acquisition, and secondary metabolisms affect virulence and fitness in Metarhizium robertsii.

Based on genomic analysis, polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways account for biosynthesis of the majority of the secondary metabolites produced by the entomopathogenic fungus Metarhizium robertsii. To evaluate the contribution of these pathways to M. robertsii fitness and/or virulence, mutants deleted for mrpptA, the Sfp-type 4' phosphopantetheinyl transferase gene required for their activation were generated. ΔmrpptA strains were deficient in PKS and NRPS activity resulting in colonies that lacked the typical green pigment and failed to produce the nonribosomal peptides (destruxins, serinocylins, and the siderophores ferricrocin and metachelins) as well as the hybrid polyketide-peptides (NG-39x) that are all produced by the wild type (WT) M. robertsii. The ΔmrpptA colonies were also auxotrophic for lysine. Two other mutant strains were generated: ΔmraarA, in which the α-aminoadipate reductase gene critical for lysine biosynthesis was disrupted, and ΔmrsidA, in which the L-ornithine N5-oxygenase gene that is critical for hydroxamate siderophore biosynthesis was disrupted. The phenotypes of these mutants were compared to those of ΔmrpptA to separate effects of the loss of lysine or siderophore production from the overall effect of losing all polyketide and non-ribosomal peptide production. Loss of lysine biosynthesis marginally increased resistance to H2O2 while it had little effect on the sensitivity to the cell wall disruptor sodium dodecyl sulfate (SDS) and no effect on sensitivity to iron deprivation. In contrast, combined loss of metachelin and ferricrocin through the inactivation of mrsidA resulted in mutants that were as hypersensitive or slightly more sensitive to H2O2, iron deprivation, and SDS, and were either identical or marginally higher in ΔmrpptA strains. In contrast to ΔmrpptA, loss of mrsidA did not completely abolish siderophore activity, which suggests the production of one or more non-hydroxamate iron-chelating compounds. Deletion of mrpptA, mrsidA, and mraarA reduced conidium production and conidia of a GFP-tagged ΔmrpptA strain displayed a longer germination delay than WT on insect cuticles, a deficiency that was rescued by lysine supplementation. Compared with WT, ΔmrpptA strains displayed ∼19-fold reduction in virulence against Drosophila suzukii. In contrast, lysine auxotrophy and loss of siderophores accounted for ∼2 and ∼6-fold decreases in virulence, respectively. Deletion of mrpptA had no significant effect on growth inhibition of Bacillus cereus. Our results suggest that PKS and NRPS metabolism plays a significant role in M. robertsii virulence, depresses conidium production, and contributes marginally to resistance to oxidative stress and iron homeostasis, but has no significant antibacterial effect.

Molecular Genetics of Secondary Chemistry in Metarhizium Fungi.

As with many microbes, entomopathogenic fungi from the genus Metarhizium produce a plethora of small molecule metabolites, often referred to as secondary metabolites. Although these intriguing compounds are a conspicuous feature of the biology of the producing fungi, their roles in pathogenicity and other interactions with their hosts and competing microbes are still not well understood. In this review, secondary metabolites that have been isolated from Metarhizium are cataloged along with the history of their discovery and structural elucidation and the salient biological activities attributed to them. Newly available genome sequences revealed an abundance of biosynthetic pathways and a capacity for producing SMs by Metarhizium species that far exceeds the known chemistry. Secondary metabolism genes identified in nine sequenced Metarhizium species are analyzed in detail and classified into distinct families based on orthology, phylogenetic analysis, and conservation of the gene organization around them. This analysis led to the identification of seven hybrid polyketide/nonribosomal peptide synthetases (M-HPNs), two inverted hybrid nonribosomal peptide/polyketide synthetases (M-IHs), 27 nonribosomal peptide synthetases (M-NRPSs), 14 nonribosomal peptide synthetase-like (M-NPL) pathways, 32 polyketide synthases, and 44 terpene biosynthetic genes having a nonuniform distribution and largely following established phylogenetic relationships within the genus Metarhzium. This systematization also identified candidate pathways for known Metarhizium chemistries and predicted the presence of unknown natural products for this genus by drawing connections between these pathways and natural products known to be produced by other fungi.

Optimization of different process variables for the production of an indolizidine alkaloid, swainsonine from Metarhizium anisopliae.

Swainsonine is a polyhydroxylated indolizidine alkaloid having anticancer, antimetastatic, antiproliferative and immunomodulatory activities and also potential therapeutic applications against AIDS. In the present study, ten isolates of M. anisopliae were screened and enzyme assayed for the production of swainsonine in different media (Complex oatmeal, Czapekdox media with and without lysine (8% w/v) and Sabouraud dextrose broth (SDB)). Among these strains, ARSEF 1724 (UM8) was found to produce highest amount of swainsonine (1.34 µg/l) after 72 h of incubation under shake flask conditions at 180 rpm and 28 °C in complex oatmeal media. In order to maximize the yield of swainsonine the media composition including macro and micronutrients were optimized. The process variables including the chemical factors like carbon sources, nitrogen sources of both organic and inorganic nature and pH with constant inoculum size (1 × 10(8) spores/ml) were screened using classical one-factor-at-a-time (OFAT) approach to find their optimum levels. The present study shows that the nutrient requirement is specific for each strain of Metarhizium. Oatmeal extract (6%) was found to be the best supporting media along with nitrogen source, glucose (2%) as best carbon source and pH (~5) as the best for swainsonine production.

Differential expression of insect and plant specific adhesin genes, Mad1 and Mad2, in Metarhizium robertsii.

Metarhizium robertsii is an entomopathogenic fungus that is also plant rhizosphere competent. Two adhesin-encoding genes, Metarhizium adhesin-like protein 1 (Mad1) and Mad2, are involved in insect pathogenesis or plant root colonization, respectively. Here we examined the differential expression of the Mad genes when grown on a variety of soluble (carbohydrates and plant root exudate) and insoluble substrates (locust, tobacco hornworm, and cockroach cuticle, chitin, tomato stems, cellulose, and starch) and during insect, Plutella xylostella, infection. On insect cuticles Mad1 was up regulated, whereas bean root exudate and tomato stems resulted in the up regulation of Mad2. During the early stages of insect infection Mad1 was expressed while Mad2 was not expressed until fungal hyphae emerged and conidiated on the insect cadaver. The regulation of Mad2 was compared to that of other stress-related genes (heat shock protein (Hsp)30, Hsp70, and starvation stress gene A (ssgA)). Mad2 was generally up regulated by nutrient starvation (similar to ssgA) but not by pH, temperature, oxidative or osmotic stresses. Whereas Hsp30 and Hsp70 were generally up regulated at 37 °C or by oxidative stress even under nutrient enriched conditions. We fused the promoter of the Mad2 gene to a marker gene (green fluorescent protein (GFP)) and confirmed that Mad2 was up regulated when M. robertsii was grown in the presence of nutrient starvation. Examination of the promoter region of Mad2 revealed that it possessed two copies of a stress-response element (STRE) known to be regulated under the general stress-response pathway.

Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum.

Metarhizium spp. are being used as environmentally friendly alternatives to chemical insecticides, as model systems for studying insect-fungus interactions, and as a resource of genes for biotechnology. We present a comparative analysis of the genome sequences of the broad-spectrum insect pathogen Metarhizium anisopliae and the acridid-specific M. acridum. Whole-genome analyses indicate that the genome structures of these two species are highly syntenic and suggest that the genus Metarhizium evolved from plant endophytes or pathogens. Both M. anisopliae and M. acridum have a strikingly larger proportion of genes encoding secreted proteins than other fungi, while ~30% of these have no functionally characterized homologs, suggesting hitherto unsuspected interactions between fungal pathogens and insects. The analysis of transposase genes provided evidence of repeat-induced point mutations occurring in M. acridum but not in M. anisopliae. With the help of pathogen-host interaction gene database, ~16% of Metarhizium genes were identified that are similar to experimentally verified genes involved in pathogenicity in other fungi, particularly plant pathogens. However, relative to M. acridum, M. anisopliae has evolved with many expanded gene families of proteases, chitinases, cytochrome P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degradation, detoxification, and toxin biosynthesis that may facilitate its ability to adapt to heterogeneous environments. Transcriptional analysis of both fungi during early infection processes provided further insights into the genes and pathways involved in infectivity and specificity. Of particular note, M. acridum transcribed distinct G-protein coupled receptors on cuticles from locusts (the natural hosts) and cockroaches, whereas M. anisopliae transcribed the same receptor on both hosts. This study will facilitate the identification of virulence genes and the development of improved biocontrol strains with customized properties.

MOS1 osmosensor of Metarhizium anisopliae is required for adaptation to insect host hemolymph.

Entomopathogenic fungi such as Metarhizium anisopliae infect insects by direct penetration of the cuticle, after which the fungus adapts to the high osmotic pressure of the hemolymph and multiplies. Here we characterize the M. anisopliae Mos1 gene and demonstrate that it encodes the osmosensor required for this process. MOS1 contains transmembrane regions and a C-terminal Src homology 3 domain similar to those of yeast osmotic adaptor proteins, and homologs of MOS1 are widely distributed in the fungal kingdom. Reverse transcription-PCR demonstrated that Mos1 is up-regulated in insect hemolymph as well as artificial media with high osmotic pressure. Transformants containing an antisense vector directed to the Mos1 mRNA depleted transcript levels by 80%. This produced selective alterations in regulation of genes involved in hyphal body formation, cell membrane stiffness, and generation of intracellular turgor pressure, suggesting that these processes are mediated by MOS1. Consistent with a role in stress responses, transcript depletion of Mos1 increased sensitivity to osmotic and oxidative stresses and to compounds that interfere with cell wall biosynthesis. It also disrupted developmental processes, including formation of appressoria and hyphal bodies. Insect bioassays confirmed that Mos1 knockdown significantly reduces virulence. Overall, our data show that M. anisopliae MOS1 mediates cellular responses to high osmotic pressure and subsequent adaptations to colonize host hemolymph.

A scorpion neurotoxin increases the potency of a fungal insecticide.

The low virulence of the insecticidal fungus Metarhizium anisopliae has stymied its widespread use in controlling insect pests. We show that high-level expression of an insect-specific neurotoxin from the scorpion Androctonus australis in hemolymph by M. anisopliae increases fungal toxicity 22-fold against tobacco hornworm (Manduca sexta) caterpillars and ninefold against adult yellow fever mosquitoes (Aedes aegypti) without compromising host specificity. Prelethal effects include reduced mobility and feeding of the insects targeted.